Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-p44/42 MAPK (Thr202/Tyr204) Sandwich ELISA Kit #7177

Kit Includes Volume Solution Color
Phospho-p44/42 MAPK (Thr202/Tyr204) Rabbit Antibody Coated Microwells 96 tests
p44/42 MAPK Mouse Detection Antibody 11 milliliters Green
Anti-mouse IgG HRP-Linked Ab 11 milliliters Red
TMB Substrate 11 milliliters Colorless
STOP Solution 11 milliliters Colorless
Sealing Tape 2 sheets
20X Wash Buffer 25 milliliters Colorless
Sample Diluent 25 milliliters Blue
Cell Lysis Buffer (10X) # 9803 15 milliliters Yellowish

Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer (10X), which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H M

Reactivity Key:  H=Human  M=Mouse

Description

CST’s PathScan® Phospho-p44/42 MAPK (Thr202/Tyr204) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of p44/42 MAPK when phosphorylated at Thr202/Tyr204 . A Phospho-p44/42 MAPK Rabbit Antibody* has been coated onto the microwells. After incubation with cell lysates, phospho-p44/42 MAPK (Thr202/Tyr204) is captured by the coated antibody. Following extensive washing, a p44/42 MAPK Mouse Detection Antibody* is added to detect the captured phospho-p44/42 MAPK protein. Anti-mouse IgG, HRP-linked antibody #7076* is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of absorbance for this developed color is proportional to the quantity of p44/42 MAPK phosphorylated at Thr202/Tyr204.*Antibodies in the kit are custom formulations specific to the kit.

Specificity / Sensitivity

CST’s PathScan® Phospho-p44/42 MAPK (Thr202/Tyr204) Sandwich ELISA Kit #7177 detects endogenous levels of phospho-p44/42 MAPK when phosphorylated at Thr202/Tyr204. As shown in Figure 1, a significant induction of p44/42 MAPK phosphorylation at Thr202/Tyr204 can be detected in PDGF-treated NIH/3T3 cells using the Phospho-p44/42 MAPK (Thr202/Tyr204) Sandwich ELISA Kit #7177. The level of total p44/42 MAPK detected by Western analysis remains unchanged. Note: This assay may have an increased absorbance reading if the diluted lysate that is applied to the microwells contains a final concentration of 0.5% sodium deoxycholate or 0.05% SDS.

Sandwich ELISA

Sandwich ELISA

Figure 1. Treatment of NIH/3T3 cells with PDGF stimulates phosphorylation of p44/42 MAPK at Thr202/Tyr204, detected by the PathScan® Phospho-p44/42 MAPK (Thr202/Tyr204) Sandwich ELISA Kit #7177, but does not affect the level of p44/42 MAPK detected by Western analysis. NIH/3T3 cells (80-90% confluent) were starved overnight and treated with 100 ng/mL PDGF for 5 minutes at 37oC. The absorbance readings at 450 nm are shown in the top figure, while the corresponding Western blots, using p44/42 MAP Kinase Antibody #9102 (left panel) and Phospho-p44/42 MAPK (Thr202/Tyr204) (D13.14.4E) Rabbit mAb #4370 (right panel), are shown in the bottom figure.

Sequence

Sequence

Figure 2. The relationship between the protein concentration of the lysate from untreated and PDGF-treated NIH/3T3 cells and the absorbance at 450 nm is shown.

Background

p44/42 MAPK (Erk1 and Erk2), SAPK/JNK and p38 MAPK function in protein kinase cascades that play a critical role in the regulation of cell growth, differentiation and control of cellular responses to cytokines and stress.p44/42 MAPK (Erk1 and Erk2) is activated by growth and neurotrophic factors. Activation occurs through phosphorylation of threonine and tyrosine residues (Thr202 and Tyr204 in human Erk1) at the sequence T*EY* by a single upstream MAP kinase kinase (MEK).SAPK/JNK and p38 MAPK are activated by inflammatory cytokines and by a wide variety of cellular stresses. Activation of SAPK/JNK occurs via phosphorylation at Thr183 and Tyr185 by the dual specificity enzyme SEK/MKK4. Both MKK3 and SEK phosphorylate p38 MAPK on tyrosine and threonine at the sequence T*GY* to activate p38 MAP kinase.

  1. Lewis, T. S. et al. (1998) Adv. Cancer Res. 74, 49-139.
  2. Garrington, T.P. and Johnson, G.L. (1999) Curr. Opin. Cell. Biol. 11, 211-218.
  3. Schaeffer, H.J. and Weber, M.J. (1999) Mol. Cell. Biol. 19, 2435-2444.
  4. Whitmarsh, A.J. and Davis, R.J. (1998) Trends Biochem. Sci. 23, 481-485.
  5. Cobb, M.H. (1999) Prog. Biophys. Mol. Biol. 71, 479-500.

Application References

Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!

Companion Products

Product Pathways

Drug Discovery Tools

Featured Technologies

Protein Classes